Abstract
BackgroundBudded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC).ResultsA rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu) in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i..ConclusionThe successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.
Highlights
Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins
The antibody-based assay allows the time reduction required for baculovirus titer, but the antibody for a baculovirus-specific protein, DNA-Binding Protein (DBP) which is expensive is mandatory
Recombinant Bombyx mori nucleopolyhedrovirus (BmNPV)-CP-/bx-GGT2 stock [15], whose titer was determined by end-point dilution method, was diluted and its baculovirus DNA was extracted from each diluted baculovirus DNA solution by High Pure Viral Nucleic Acid Kit
Summary
Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. We tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. Budded baculoviruses are applied for displaying the recombinant proteins on their surface for antibody production, functional analysis of receptors and vaccine production [5,6,7]. In order to use baculoviruses in gene therapy, the development of efficient production process towards high-titer preparation is required, because of low baculovirus transduction efficiency in mammalian cells compared to other viral delivery system, e.g. retroviruses
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